ω-3 PUFA for Secondary Prevention of White Matter Lesions and Neuronal Integrity Breakdown in Older Adults

Key Points Question Does ω-3 polyunsaturated fatty acid treatment reduce cerebral white matter lesion (WML) accumulation and neuronal integrity breakdown among older adults? Findings In this randomized clinical trial of 102 participants, ω-3 treatment failed to significantly reduce WML progression and neuronal integrity breakdown among all participants; however, apolipoprotein E ε4 allele (APOE*E4) carriers who received ω-3 had significant reductions in neuronal integrity breakdown over 3 years. Meaning Although ω-3 treatment failed to reach significant reduction in WML progression and neuronal integrity breakdown among all participants at risk for dementia, the findings suggest that APOE*E4 carriers may benefit from ω-3 treatment.


Introduction
Cerebral white matter lesion (WML) accumulation, identified as hyperintensities on magnetic resonance imaging (MRI) fluid-attenuated inversion recovery (FLAIR) scans, are thought to play a major role in the development of cognitive decline and dementia, including Alzheimer disease (AD). 1,2[17] Diet-derived bioactive lipids, such as the ω-3 polyunsaturated fatty acids (PUFAs) eicosapentaenoic acid (EPA; 20:5) and docosahexaenoic acid (DHA; 22:6), which are found in deep, cold-water fish, 18 and the plasma concentrations that manifest from their consumption are associated with reduced WML burden. 19,20These ω-3 PUFAs are modifiable structural phospholipids that may alter cell signaling and metabolism toward a less inflammatory state by acting as substrate for synthesis of lipophilic inflammation-resolving metabolites 21 ; ω-3 has been shown to reduce microglial cytokine release (ie, tumor necrosis factor--α) and endothelial and immune cell surface protein CD54 expression. 22,23CD54 protein encoding inferred by the intercellular adhesion molecule-1 (ICAM-1) gene locus can increase blood-brain barrier permeability. 24Meta-analyses of ω-3 clinical trials have shown that it can downregulate soluble adhesion molecules, specifically ICAM-1, although other ω-3 trials have identified modest effects on cerebral perfusion and white matter integrity parameters. 22,25,26Plasma ω-3 explains 24% of the WML variation, and higher levels are associated with a 40% reduction in probability for WML among older adults. 19,20Plasma ω-3 concentrations above 11.0 mg/dL (to convert to millimoles per liter, multiply by 0.0355) are associated with less WML-mediated executive cognitive function decline in older adults without dementia, 27 and 1.65 g per day of ω-3 has been shown clinically to supersede this hypothetical neuroprotective threshold in mild-to-moderate AD.Consequently, we targeted enrollment to a population of older adults harboring WMLs and suboptimal ω-3 status to determine whether ω-3 treatment prevents WML progression and neuronal integrity breakdown over 3 years vs a placebo.

Study Design and Participant Enrollment
The Polyunsaturated Fatty Acids for the Prevention of Cerebral Small Vessel Disease and Inflammation in Aging (PUFA trial) protocol and baseline results are available elsewhere. 28The trial protocol and statistical analysis plan are shown in Supplement 1.Briefly, this 3-year, quadrupleblinded, placebo-controlled, randomized clinical trial was conducted at Oregon Health & Science University (OHSU) and compared ω-3 vs placebo treatment (1:1) in participants recruited from the Portland, Oregon, metropolitan area.Eligibility requirements included age (Ն75 years) and being cognitively intact or with mild cognitive impairment, defined as Clinical Dementia Rating (CDR) of 0.5 or less (range, 0.0-0.5, with lower scores denoting cognitively and functionally intact) and Mini-Mental State Examination (MMSE) score above 23 (range, 24-30, with higher scores denoting better cognitive function).Brain MRI screen used a 1-mm isotropic FLAIR algorithm to ascertain total WML eligibility of 5 cm 3 or more.Plasma or whole-blood spot ω-3 eligibility excluded candidates with objective evidence of ω-3 status already above the threshold associated with neuroprotection (Ն11.0 mg/dL or Ն5.5 weight percentage of total fatty acids) (eTable 1 in Supplement 2). 27Race and ethnicity were self-reported by the participants and are included in this study to reflect the generalizability of the results.

Standard Protocol Approval and Patient Consent
This randomized clinical trial follows the Consolidated Standards of Reporting Trials (CONSORT) and Good Clinical Practice guidelines, with local ethical committee approval (OHSU Institutional Review Board).All participants signed written informed consent.A designated independent Data and Safety Monitoring Board was established and met biannually to review progress.

Randomization and Masking
Eligible consented participants were randomized by an independent statistician (C.F.M.) using a modified minimization algorithm to ω-3 or placebo soft gels balanced by age across 5-year block segments, sex, education (up to high school graduate, at least some college, and baccalaureate graduate or postgraduate), screening total WML volume, and CDR scores. 29Participants, study investigators, research staff, and health care practitioners were masked to treatment assignments.
Study ω-3 and placebo soft gels were independently received, processed, and distributed by OHSU research pharmacy staff.Blinding integrity was assessed with a short questionnaire completed by the participants and the investigators. 30

Procedures and Intervention
As described previously, 28 eligibility was determined over 2 sequential clinic visits lasting 5 hours.MMSE, CDR, geriatric depression scale, safety laboratory (eg, comprehensive metabolic panel and complete blood counts), and ω-3 status (plasma or whole-blood spot) were determined at visit 1.
Screening MRI for WML eligibility was at visit 2. Participants underwent a general and neurological examination, neuropsychological testing, safety laboratory workup, and other blood draws systematically across the trial visits. 28Treatment compliance was recorded through pill counts at each clinic visit, and telephone contact every 3 months between the clinic visits was performed to review compliance.Pill counts less than 80% adherent triggered further contact to resolve compliance.Mass-mailers and subsequent telephone screenings of 1100 people identified 299 candidates for invitation to an in-clinic assessment of general and neurological signs and symptoms, cognition, blood ω-3 status, and WMLs.The first clinic visit including ω-3 status yielded 233 candidates for WML MRI screening.There were 124 of the 233 who failed to meet WML screening eligibility (53%), and another individual with poor blood pressure management, which left 108 eligible individuals.The active group received 3 soft gels daily yielding 1.65 g of ω-3 (975 mg of EPA and 675 mg of DHA) in a triacylglycerol emulsion, whereas the placebo group received 3 soybean oil JAMA Network Open | Neurology ω-3 PUFA for Prevention of White Matter Lesions in Older Adults soft gels daily indistinguishable from active treatment according to taste, appearance, smell and texture by the manufacturer (Nordic Naturals, Inc).Random batches of the natural product underwent systematic independent laboratory testing over the course of the trial to ensure product integrity (eTable 2 in Supplement 2).

Main Outcomes
The primary outcome was annual WML progression.Secondary outcomes were DTI-FA, medial temporal lobe gray matter (GM), and total brain GM.Treatment stratification by APOE genotype was prespecified.Baseline brain MRI was captured using a 3-T MRI instrument (Total Imaging Matrix Trio; Siemens) with phased array body transmit radiofrequency (RF) coil and a 32-channel head RF coil receiver housed in the OHSU Advanced Imaging Research Center.The Total Imaging Matrix Trio scanner was upgraded to a 3-T Prisma (Siemens) during the study.The RF receiver coil and sequence parameters were consistent across the upgrade, and the Prisma system was used for 32 of the 87 participants with a 1-year follow-up visit and each scan from there forward.Anatomical imaging sequences were similar to the Alzheimer Disease Neuroimaging Initiative protocols. 31Structural MRI included T 1 magnetization prepared rapid gradient echo (MPRAGE), FLAIR, pseudo-continuous arterial spin labeling, DTI, proton density-T 2 , and functional MRI with the imaging session requiring approximately 60 minutes in total: 3-dimensional (3D) T 1 -MPRAGE (orientation, axial; repetition time, 2300 ms; echo time, 3.45 ms; slice thickness, 1 mm; slices, 144; native resolution, 1 mm isotropic; acquisition matrix, 256 × 192), 3D FLAIR (orientation, sagittal; repetition time, 6000 ms; echo time, 388 ms; slice thickness, 1 mm; slices, 160; native resolution, 0.5 × 0.5 × 1 mm; acquisition matrix, 512 × 512), and DTI (orientation, axial; repetition time, 9100 ms; echo time, 88 ms; native resolution, 2 mm isotropic; slice thickness, 2 mm; slices, 72; acquisition matrix, 128 × 128; b = 0, 1000 seconds/mm 2 ).Briefly, GM volume of the medial temporal lobe, total brain, and ventricular cerebral spinal fluid volumes were segmented with FreeSurfer software version 6.0 (Harvard University), visually inspected, and manually corrected for errors.For FLAIR-derived WMLs, a semiautomated intensity-based thresholding algorithm using resampled 1 mm isotropic resolution images was used. 28,32FLAIR images were linearly coregistered to T1 images, and the mean FLAIR signal intensity in the WM was calculated.Voxel clusters at least 2.5 SD above the mean WM FLAIR signal intensity were used as seeds.The mean signal intensity of each cluster was calculated and compared with all surrounding voxels.Adjacent voxels of at least 95% of the cluster mean signal intensity were added to the cluster, and the process was repeated iteratively.The final WML mask was visually inspected and manually corrected before final volume calculation.After study completion, updated neuroimaging processing algorithms included analysis of higher resolution sequences (FLAIR, in-plane resolution 0.5 mm).Mean skeletal DTI-FA and mean, radial, and axial diffusivity were calculated using tract-based spatial statistics. 33Cognitive changes were not powered adequately but collected rather comprehensively to power future efforts (eAppendix in Supplement 2).

ω-3 PUFA and APOE Genotype
Screening for study inclusion required either Holman Omega-3 whole-blood spot test EPA plus DHA less than 5.5% total fatty acids, which allows for a 10-day result turnaround, or plasma EPA plus DHA less than 11.0 mg/dL, which requires 14-to 20-day turnaround. 34,35Plasma fatty acid concentrations were analyzed by gas chromatography with flame ionization detection as previously described. 36tty acids were identified by comparison with a standard mixture of fatty acids (GLC OQ-A; NuCheck Prep), which was also used to determine individual fatty acid calibration curves.The C23:0 TG was used to calculate recovery efficiency of the assay and applied to all fatty acids.Plasma fatty acids were expressed as absolute concentrations (eg, micrograms per milliliter of plasma).APOE genotype was determined by polymerase chain reaction test.

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ω-3 PUFA for Prevention of White Matter Lesions in Older Adults

Safety
Safety evaluation included general and neurological examination, heart rate and blood pressure, use of concomitant medications and dietary supplements, and blood laboratory testing (ie, complete blood counts, comprehensive metabolic panel, prothrombin time, and international normalized ratio) performed at screening and at 6, 12, 24, and 36 months. 28Following all MRI assessments, T1 and FLAIR images were reviewed for incidental findings of relevance (eg, cortical infarct, tumor, or normal pressure hydrocephalus), and participants with findings were notified within 1 week by telephone for a discussion.A primary care visit was encouraged within 21 days of any remarkable findings with the research study MRI provided to facilitate expedited care as deemed necessary.
Adverse events (AEs) were monitored in real time with weekly staff review using clinical judgment and reports to the local institutional review board and an external data and safety monitoring board.
The data and safety monitoring board convened biannually for updates on the progress and the review of study data including any AE coded by organ system under Medical Dictionary for Regulatory Activities format.Participants who withdrew from the study received an early termination visit either in person or by telephone to document withdrawal explanation and abbreviated follow-up plan for off-study treatment.

Statistical Analysis
Data analysis was performed from February 2020 to July 2022.Statistical power and sample size were informed by the longstanding Oregon Brain Aging Study (OBAS) cohort of participants aged 80 years and older with CDR less than or equal to 0.5 and WML greater than or equal to 6 cm 3 , which was prevalent in 80% of the OBAS cohort.Simulation analysis estimated that the current trial placebo group should accumulate a mean (SD) of 7 (5) cm 3 of WML over 3 years.However, during recruitment efforts, the prevalence of WML greater than or equal to 6 cm 3 was 46.7% (109 of 233 participants), and documented protocol modifications were made to meet enrollment without sacrificing the primary hypotheses.Therefore, the threshold for WML inclusion was lowered to 5 cm 3 , age to 75 years or older, and sample size from 150 to 100 participants.Together, these modifications sustained 80% power to test the hypothesis that ω-3 attenuates annual WML progression by 50% compared with the placebo (ie, mean [SD], 3.5 [5.0] cm 3 over 3 years in ω-3 group vs 7.0 [5.0] cm 3 in the placebo group).Considering 30% attrition over 3 years, a total of 94 individuals were required, with 33 completers per treatment group.
Differences in annual change among study outcomes by treatment group were assessed using linear mixed-effects models, as described elsewhere, 28 using R statistical software version 4.2 (R Project for Statistical Computing), 37 with lme4, 38 nlme, 39 and ggplot2 40 plot packages. 28Treatment stratification by APOE genotype was prespecified.Interaction terms between treatment and time evaluated annualized change differences by group.Model covariates included baseline age, sex, history of vascular disease (yes/no, indicating any one of the following: history of cardiac arrest, angina pectoris, angioplasty, cardiac bypass surgery, congestive heart failure, and atrial fibrillation), history of hypertension (yes/no), history of depression (yes/no), and MRI system used.MRI system was included as a covariate for the volumetric and diffusion MRI outcomes to account for system upgrade during the trial and MRI volumes (eg, WML, total brain volume, medial temporal lobe volume, and ventricular volume) were adjusted for intracranial volume at each MRI visit.Estimates and 95% CIs for WML were calculated on the original scale of cubic centimeters using linear mixedeffects models (ie, using pretransformed WML as outcomes) to provide crude annual volume change and same covariate corrections.Adverse events were analyzed using Fisher exact test in all randomized participants who received treatment.All hypothesis testing was 2-sided with results considered statistically significant at P < .05.Model fitness and confounding evaluations are provided in the eAppendix in Supplement 2.

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ω-3 PUFA for Prevention of White Matter Lesions in Older Adults

Enrollment, Demographics, and Attrition
Recruitment was initiated in May of 2014 and concluded in July 2016.The participants' final visit was in September 2019.Figure 1 presents the participant enrollment and flow through the trial beginning with mass mailers and telephone screens, yielding 299 potential candidates for in-clinic screenings, of whom 102 individuals (62 women [60.8%]; mean age, 81 years [range, 75-96 years]) met inclusion criteria for both WML greater than or equal to 5 cm 3 and plasma ω-3 PUFA less than 11.0 mg/dL or less than 5.5% whole-blood spot and agreed to be randomized (1:1; 51 per group).Twenty-eight participants (28%) carried an APOE*E4 genetic allele.The mean (SD) MMSE score was 28.0 (1.7), and the mean (SD) score MoCA was 24.0 (3.2), with all participants considered to not have dementia (eg, MMSE >23 and CDR of Յ0.5) (Table 1).Ninety participants completed the 1-year visit (44 in the ω-3 group and 46 in the placebo group), 81 completed the 2-year visit (40 in the ω-3 group and 41 in the placebo), and 78 completed the final 3-year visit (39 per group).Forty-five participants in the ω-3 PUFA group and 42 in the placebo group had at least 1 follow-up MRI to calculate change by treatment group under the intention-to-treat (ITT) protocol.The annual rate of participant attrition   a The intention-to-treat (ITT) analysis includes all randomly assigned participants with at least 1 follow-up magnetic resonance imaging scan (n = 87).

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ω-3 PUFA for Prevention of White Matter Lesions in Older Adults over the 3-year duration was similar among the treatment groups (8.5%; P for difference = .32,Fisher exact test).As expected, much of the attrition occurred earlier in the trial (11.7% by the end of year 1, 20.5% by the end of year 2, and 23.5% by the end of year 3).

Safety and Tolerability
Serious AEs (SAEs) or AEs between groups did not differ (Table 3).SAEs occurred in 16 participants (31.4%) across both groups (hospitalizations, P = .82;deaths, P = .36;Fisher exact test for difference between groups).Hospitalization prevalence was 25.4% (26 participants) over the course of the study (12 participants in the ω-3 group and 14 participants in the placebo group).Five deaths were recorded, including 4 in the ω-3 group: 1 due to a fatal fall, 1 renal and urinary tract disorder, 1 hematologic vascular disorder, and 1 prostate cancer metastasis.The 1 death in the placebo group was due to a nonhematological vascular disorder.No SAEs were deemed as attributable to the a The intention-to-treat cohort includes all randomly assigned participants with at least 1 follow-up magnetic resonance imaging scan (n = 87).
b Annualized changes were calculated from primary mixed-effects models as time trajectories for ω-3 and placebo groups.
c Linear response difference was taken as the treatment-time interaction coefficient from the primary mixed-effects models.
d Omega-3 treatment effect sizes were taken as the t-statistic from the primary mixedeffects model for the treatment-time interaction.
e Cohen d was calculated from the primary mixed-effects models using t-statistic and appropriate df for each outcome.The intention-to-treat protocol includes all 87 randomized participants with at least 1 follow-up magnetic resonance imaging (MRI) scan.The mean annual periventricular WML increase was 0.09 cm 3 (95% CI, 0.05 to 0.13 cm 3 ) in the ω-3 group and 0.11 cm 3 (95% CI, 0.07 to 0.15 cm 3 ) in the placebo group (P = .28).The mean annual increase in subcortical WML was 0.03 cm 3 (95% CI, −0.04 to 0.09 cm 3 ) in the ω-3 group and 0.04 cm 3 (95% CI, −0.02 to 0.11 cm 3 ) in the placebo group (P = .72).Mean annual DTI radial diffusivity increase was 3.143 × 10

Figure
Figure 1.Participant Enrollment Flowchart indicates polyunsaturated fatty acid.
-3 PUFA for Prevention of White Matter Lesions in Older Adults ω 1. Participant Enrollment Flowchart

Table 1 .
Baseline Demographic and Clinical Characteristics a b Body mass index is calculated as weight in kilograms divided by height in meters squared.JAMA Network Open | Neurologyω-3 PUFA for Prevention of White Matter Lesions in Older Adults ω-3 PUFA

Table 2 .
ω-3 Polyunsaturated Fatty Acid Effects on the Main Outcomes in All Participants and by APOE*E4 Carrier Status Over 3 Years Under the Intention to Treat Protocol a Open | Neurology ω-3 PUFA for Prevention of White Matter Lesions in Older Adults Downloaded from jamanetwork.combyguest on 08/04/2024treatments. AEs occurred in 44 participants in the ω-3 group and 41 in the placebo group (P for difference = .78,Fisher exact test).The most common AEs were injurious falls (30.3%; 17 in the ω-3 group and 14 in the placebo group), musculoskeletal and connective tissue disorders (25.4%, predominantly preexisting joint conditions, such as osteoarthritis), and gastrointestinal disorders (24.5%; 10 participants in the ω-3 group and 15 participants in the placebo group; predominantly stomach pain, loose stool, gas, or flatulence).Twelve participants in the ω-3 and placebo groups Figure 2. Effects of ω-3 Polyunsaturated Fatty Acids on White Matter Lesion (WML) and Neuronal Integrity Breakdown in All Participants and by Apolipoprotein E Genotype

Effects on Exploratory Outcomes Regional WML, DTI-Radial, Mean, and Axial Diffusivity, and Executive Cognitive z-Score
discontinued study treatment due mainly to AEs classified as unrelated to the experimental treatments.There was no dropout difference due to SAEs or AEs among the groups (7 participants).The dropout rates due to AEs were 11.7% (6 participants) in the ω-3 group and 5.9% in the placebo group (3 participants) (P for difference = .49,Fisher exact test).

Table 3 .
Adverse Events by Experimental Group -3 PUFA for Prevention of White Matter Lesions in Older Adults a Adverse events are defined according to the Medical Dictionary for Regulatory Activities terminology.Rates between groups across categories were similar (Fisher exact test, P = .60).b Fisher exact test, P = .82. c Fisher exact test, P = .36.JAMA Network Open | Neurologyω